No description available.

A selection system for yeast cells expressing mutated invertase (EC 3.2.1.26, SUC2) with altered fructotransferase activity was developed based on the survival of a fructose-intolerant strain in the presence of suitable acceptor substrates and sucrose. Cells of such a strain expressing a wild-type hydrolase activity will not grow due to the release of free fructose from sucrose. Cells expressing an inactive invertase mutant will not grow since they cannot cleave the sucrose, the sole carbon source. Only cells expressing sucrose fructosyl-transferase activity will thrive, growing on the released glucose, the fructosyl moiety not being released. A strain of Saccharomyces cerevisiae was engineered to be intolerant of the presence of fructose in its growth media. This was achieved by inducing a condition in yeast similar to liver cells of humans suffering from hereditary fructose intolerance (MIM 22960). This disorder results from a deficiency of aldolase B (EC 4.1.2.13), and phosphorylation of fructose by ketohexokinase (EC 2.7.1.3) results in an accumulation of fructose 1-phosphate, with a consequent depletion of cytoplasmic phosphate and ATP. Thus, cells in which ketohexokinase phosphorylates fructose, but which lack aldolase B, are intolerant of fructose. Yeast possess neither of these enzymes, and so expression of ketohexokinase in yeast would result in fructose-intolerance. A strain of yeast, for ketohexokinase expression, was initially bred to be unable to metabolize sucrose or fructose, yet remain capable of utilizing glucose, as well as lacking non-specific phosphatases, to prevent remobilization of sequestered fructose 1-phosphate. Rat liver ketohexokinase was purified to heterogeneity, and the partial amino acid sequence subsequently generated exploited to amplify a region of the ketohexokinase cDNA by PCR. This was used to probe a cDNA library, yielding clones encoding the entire ketohexokinase coding region. This was cloned into pMA91, and subsequent expression in yeast resulted in a strain intolerant of fructose in its growth medium, although still capable of growing on glucose. In order to produce a stable fructose-intolerant selection strain, a vector (pIADl) was constructed that allowed multiple integration of an expression cassette containing ketohexokinase cDNA into the rDNA locus of yeast chromosome XII. Expression of wild type invertase from the episomal plasmid pIAD3 in this strain resulted in sucrose-intolerance. A preliminary programme of mutagenesis of the SUC2 gene yielded eight libraries of about one hundred clones each. None of these contained any mutants showing solely sucrose fructosyl-transferase activity, although this system would clearly provide an ideal selection for such mutants from a much larger library.

No description available.

Thesis (Ph. D.)--University of Nevada, Reno, 2007. / "May 2007." Includes bibliographical references (leaves 75-86). Online version available on the World Wide Web.

Thesis (Ph. D.)--University of Toronto, 2004. / Adviser: Richard Glazier.

Diss. (sammanfattning) Stockholm : Karol. inst. / Titel på diss.-titelbl.: Islet glucose metabolism and insulin secretion in two animal models of glucose intolerance. Härtill 5 uppsatser.

Thesis (M.A.)--University of North Carolina at Chapel Hill, 2008. / Title from electronic title page (viewed Jun. 26, 2009). "... in partial fulfillment of the requirements for the degree of Master of Arts in the Department of Political Science, Concentration TransAtlantic Studies." Discipline: Political Science; Department/School: Political Science.

Dissertation (Ph.D.)--University of Toledo, 2009. / Typescript. "Submitted to the Graduate Faculty as partial fulfillment of the requirements for The Doctor of Philosophy Degree in Higher Education." Bibliography: leaves 274-302.

Thesis (M.A.)--East Tennessee State University, 2001. / Title from electronic submission form. Includes bibliographical references. Also available via Internet at the UMI web site.

Thesis (doctoral)--Lund, 1982.